Background and aims Bacterial Non-Specific Acid Phosphatase (NSAP) enzymes are capable of dephosphorylating diverse organic phosphoesters but are rarely studied: their distribution in natural and ...managed environments is poorly understood. The aim of this study was to generate new insight into the environmental distribution of NSAPs and establish their potential global relevance to cycling of organic phosphorus. Methods We employed bioinformatic tools to determine NSAP diversity and subcellular localization in microbial genomes; used the corresponding NSAP gene sequences to census metagenomes from diverse ecosystems; studied the effect of long-term land management upon NSAP diversity and abundance. Results Periplasmic class B NSAPs are poorly represented in marine and terrestrial environments, reflecting their association with enteric and pathogenic bacteria. Periplasmic class A and outer membrane-associated class C NSAPs are cosmopolitan. NSAPs are more abundant in marine than terrestrial ecosystems and class C more abundant than class A genes, except in an acidic peat where class A genes dominate. A clear effect of land management upon gene abundance was identified. Conclusions NSAP genes are cosmopolitan. Class C genes are more widely distributed: their association with the outer-membrane of cells gives them a clear role in the cycling of organic phosphorus, particularly in soils.
Activating somatic mutations of the tyrosine kinase domain of epidermal growth factor receptor (EGFR) have recently been characterized in a subset of patients with advanced non-small cell lung cancer ...(NSCLC). Patients harboring these mutations in their tumors show excellent response to EGFR tyrosine kinase inhibitors (EGFR-TKIs). The EGFR-TKI gefitinib has been approved in Europe for the treatment of adult patients with locally advanced or metastatic NSCLC with activating mutations of the EGFR TK. Because EGFR mutation testing is not yet well established across Europe, biomarker-directed therapy only slowly emerges for the subset of NSCLC patients most likely to benefit: those with EGFR mutations.
The “EGFR testing in NSCLC: from biology to clinical practice” International Association for the Study of Lung Cancer-European Thoracic Oncology Platform multidisciplinary workshop aimed at facilitating the implementation of EGFR mutation testing. Recommendations for high-quality EGFR mutation testing were formulated based on the opinion of the workshop expert group.
Co-operation and communication flow between the various disciplines was considered to be of most importance. Participants agreed that the decision to request EGFR mutation testing should be made by the treating physician, and results should be available within 7 working days. There was agreement on the importance of appropriate sampling techniques and the necessity for the standardization of tumor specimen handling including fixation. Although there was no consensus on which laboratory test should be preferred for clinical decision making, all stressed the importance of standardization and validation of these tests.
The recommendations of the workshop will help implement EGFR mutation testing in Europe and, thereby, optimize the use of EGFR-TKIs in clinical practice.
Small cell lung cancer (SCLC) has recently been subcategorized into neuroendocrine (NE)‐high and NE‐low subtypes showing ‘immune desert’ and ‘immune oasis’ phenotypes, respectively. Here, we aimed to ...characterize the tumor microenvironment according to immune checkpoints and NE subtypes in human SCLC tissue samples at the protein level. In this cross‐sectional study, we included 32 primary tumors and matched lymph node (LN) metastases of resected early‐stage, histologically confirmed SCLC patients, which were previously clustered into NE subtypes using NE‐associated key RNA genes. Immunohistochemistry (IHC) was performed on formalin‐fixed paraffin‐embedded TMAs with antibodies against CD45, CD3, CD8, MHCII, TIM3, immune checkpoint poliovirus receptor (PVR), and indoleamine 2,3‐dioxygenase (IDO). The stroma was significantly more infiltrated by immune cells both in primary tumors and in LN metastases compared to tumor nests. Immune cell (CD45+ cell) density was significantly higher in tumor nests (P = 0.019), with increased CD8+ effector T‐cell infiltration (P = 0.003) in NE‐low vs NE‐high tumors. The expression of IDO was confirmed on stromal and endothelial cells and was positively correlated with higher immune cell density both in primary tumors and in LN metastases, regardless of the NE pattern. Expression of IDO and PVR in tumor nests was significantly higher in NE‐low primary tumors (vs NE‐high, P < 0.05). We also found significantly higher MHC II expression by malignant cells in NE‐low (vs NE‐high, P = 0.004) tumors. TIM3 expression was significantly increased in NE‐low (vs NE‐high, P < 0.05) tumors and in LN metastases (vs primary tumors, P < 0.05). To our knowledge, this is the first human study that demonstrates in situ that NE‐low SCLCs are associated with increased immune cell infiltration compared to NE‐high tumors. PVR, IDO, MHCII, and TIM3 are emerging checkpoints in SCLC, with increased expression in the NE‐low subtype, providing key insight for further prospective studies on potential biomarkers and targets for SCLC immunotherapies.
Small cell lung cancer (SCLC) has recently been subcategorized into neuroendocrine (NE)‐high and NE‐low subtypes. This study demonstrates that NE‐low SCLCs are associated with increased immune‐cell infiltration compared to NE‐high tumours. We found expression of PVR, indoleamine 2,3‐dioxygenase, MHCII, and TIM3 to be increased in the NE‐low subtype, highlighting these molecules as potential biomarkers and targets for SCLC immunotherapies.
Soil microbial communities contain the highest level of prokaryotic diversity of any environment, and metagenomic approaches involving the extraction of DNA from soil can improve our access to these ...communities. Most analyses of soil biodiversity and function assume that the DNA extracted represents the microbial community in the soil, but subsequent interpretations are limited by the DNA recovered from the soil. Unfortunately, extraction methods do not provide a uniform and unbiased subsample of metagenomic DNA, and as a consequence, accurate species distributions cannot be determined. Moreover, any bias will propagate errors in estimations of overall microbial diversity and may exclude some microbial classes from study and exploitation. To improve metagenomic approaches, investigate DNA extraction biases, and provide tools for assessing the relative abundances of different groups, we explored the biodiversity of the accessible community DNA by fractioning the metagenomic DNA as a function of (i) vertical soil sampling, (ii) density gradients (cell separation), (iii) cell lysis stringency, and (iv) DNA fragment size distribution. Each fraction had a unique genetic diversity, with different predominant and rare species (based on ribosomal intergenic spacer analysis RISA fingerprinting and phylochips). All fractions contributed to the number of bacterial groups uncovered in the metagenome, thus increasing the DNA pool for further applications. Indeed, we were able to access a more genetically diverse proportion of the metagenome (a gain of more than 80% compared to the best single extraction method), limit the predominance of a few genomes, and increase the species richness per sequencing effort. This work stresses the difference between extracted DNA pools and the currently inaccessible complete soil metagenome.
Mutations in the epidermal growth factor receptor gene (EGFR) represent one of the most frequent “actionable” alterations in non–small cell lung cancer (NSCLC). Typified by high response rates to ...targeted therapies, EGFR tyrosine kinase inhibitors (TKIs) are now established first-line treatment options and have transformed the treatment paradigm for NSCLC. With the recent breakthrough designation and approval of the third-generation EGFR TKI osimertinib, available systemic and local treatment options have expanded, requiring new clinical algorithms that take into account individual patient molecular and clinical profiles. In this International Association for the Study of Lung Cancer commissioned consensus statement, key pathologic, diagnostic, and therapeutic considerations, such as optimal choice of EGFR TKI and management of brain metastasis, are discussed. In addition, recommendations are made for clinical guidelines and research priorities, such as the role of repeat biopsies and use of circulating free DNA for molecular studies. With the rapid pace of progress in treating EGFR-mutant NSCLC, this statement provides a state-of-the-art review of the contemporary issues in managing this unique subgroup of patients.
Background: Gefitinib is a selective inhibitor of the epidermal growth factor (EGFR) tyrosine kinase, which is overexpressed in many cancers, including non–small-cell lung cancer (NSCLC). We carried ...out a clinical study to compare the relationship between EGFR gene copy number, EGFR protein expression, EGFR mutations, and Akt activation status as predictive markers for gefitinib therapy in advanced NSCLC. Methods: Tumors from 102 NSCLC patients treated daily with 250 mg of gefitinib were evaluated for EGFR status by fluorescence in situ hybridization (FISH), DNA sequencing, and immunohistochemistry and for Akt activation status (phospho-Akt P-Akt) by immunohistochemistry. Time to progression, overall survival, and 95% confidence intervals (CIs) were calculated and evaluated by the Kaplan–Meier method; groups were compared using the log-rank test. Risk factors associated with survival were evaluated using Cox proportional hazards regression modeling and multivariable analysis. All statistical tests were two-sided. Results: Amplification or high polysomy of the EGFR gene (seen in 33 of 102 patients) and high protein expression (seen in 58 of 98 patients) were statistically significantly associated with better response (36% versus 3%, mean difference = 34%, 95% CI = 16.6 to 50.3; P<.001), disease control rate (67% versus 26%, mean difference = 40.6%, 95% CI = 21.5 to 59.7; P<.001), time to progression (9.0 versus 2.5 months, mean difference = 6.5 months, 95% CI = 2.8 to 10.3; P<.001), and survival (18.7 versus 7.0 months, mean difference = 11.7 months, 95% CI = 2.1 to 21.4; P = .03). EGFR mutations (seen in 15 of 89 patients) were also statistically significantly related to response and time to progression, but the association with survival was not statistically significant, and 40% of the patients with mutation had progressive disease. In multivariable analysis, only high EGFR gene copy number remained statistically significantly associated with better survival (hazard ratio = 0.44, 95% CI = 0.23 to 0.82). Independent of EGFR assessment method, EGFR+/P-Akt+ patients had a statistically significantly better outcome than EGFR−, P-Akt−, or EGFR+/P-Akt− patients. Conclusions: High EGFR gene copy number identified by FISH may be an effective molecular predictor for gefitinib efficacy in advanced NSCLC.
The soil ecosystem is critical for human health, affecting aspects of the environment from key agricultural and edaphic parameters to critical influence on climate change. Soil has more unknown ...biodiversity than any other ecosystem. We have applied diverse DNA extraction methods coupled with high throughput pyrosequencing to explore 4.88 × 10(9) bp of metagenomic sequence data from the longest continually studied soil environment (Park Grass experiment at Rothamsted Research in the UK). Results emphasize important DNA extraction biases and unexpectedly low seasonal and vertical soil metagenomic functional class variations. Clustering-based subsystems and carbohydrate metabolism had the largest quantity of annotated reads assigned although <50% of reads were assigned at an E value cutoff of 10(-5). In addition, with the more detailed subsystems, cAMP signaling in bacteria (3.24±0.27% of the annotated reads) and the Ton and Tol transport systems (1.69±0.11%) were relatively highly represented. The most highly represented genome from the database was that for a Bradyrhizobium species. The metagenomic variance created by integrating natural and methodological fluctuations represents a global picture of the Rothamsted soil metagenome that can be used for specific questions and future inter-environmental metagenomic comparisons. However, only 1% of annotated sequences correspond to already sequenced genomes at 96% similarity and E values of <10(-5), thus, considerable genomic reconstructions efforts still have to be performed.
Histone deacetylase inhibitors (HDACis) have been shown to overcome resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) linked to epigenetic changes and ...epithelial-mesenchymal transition (EMT) state. This randomized phase II study evaluated the outcome of erlotinib with and without the isoform selective HDACi, entinostat.
Previously treated patients with stage IIIB/IV non-small-cell lung cancer, no prior EGFR-TKIs, and performance status ≤ 2 were randomly administered erlotinib 150 mg on days 1 through 28 plus entinostat 10 mg orally on days 1 and 15 every 28 days (EE) or erlotinib plus placebo (EP). The primary end point was 4-month progression-free survival (PFS) rate with additional end points including 6-month PFS rate, PFS, and overall survival (OS). Exploratory analyses included EMT- and EGFR-related biomarker analysis on archival tissue.
One hundred thirty-two patients were enrolled (EE, 67; EP, 65). The 4-month PFS rate was comparable for both groups (EE, 18% v EP, 20%; P = .7). In the subset of patients with high E-cadherin levels, OS was longer in the EE group compared with the EP group (9.4 v 5.4 months; hazard ratio, 0.35; 95% CI, 0.13 to 0.92; P = .03) with a corresponding trend toward increased PFS. The adverse event (AE) profile was acceptable, with rash, fatigue, diarrhea, and nausea the most common AEs in both groups.
Erlotinib combined with entinostat did not improve the outcomes of patients in the overall study population when compared with erlotinib monotherapy. High E-cadherin expression levels at time of diagnosis indicate an increased sensitivity to HDACi/EGFR-TKI inhibition providing the basis for a biomarker-driven validation study.
Background.
The U.S. Food and Drug Administration‐approved method for detecting EML4‐ALK rearrangement is fluorescence in situ hybridization (FISH); however, data supporting the use of ...immunohistochemistry (IHC) for that purpose are accumulating. Previous studies that compared FISH and IHC considered FISH the gold standard, but none compared data with the results of next‐generation sequencing (NGS) analysis.
Materials and Methods.
We studied FISH and IHC (D5F3 antibody) systematically for EML4‐ALK rearrangement in 51 lung adenocarcinoma patients, followed by NGS in case of discordance.
Results.
Of 51 patients, 4 were positive with FISH (7.8%), and 8 were positive with IHC (15.7%). Three were positive with both. NGS confirmed that four of the five patients who were positive with IHC and negative with FISH were positive for ALK. Two were treated by crizotinib, with progression‐free survival of 18 and 6 months. Considering NGS as the most accurate test, the sensitivity and specificity were 42.9% and 97.7%, respectively, for FISH and 100% and 97.7%, respectively, for IHC.
Conclusion.
The FISH‐based method of detecting EML4‐ALK rearrangement in lung cancer may miss a significant number of patients who could benefit from targeted ALK therapy. Screening for EML4‐ALK rearrangement by IHC should be strongly considered, and NGS is recommended in borderline cases. Two patients who were negative with FISH and positive with IHC were treated with crizotinib and responded to therapy.
The U.S. Food and Drug Administration‐approved method for detecting EML4‐ALK rearrangement in lung cancer is fluorescence in situ hybridization (FISH); however, FISH may miss a significant number of patients who could benefit from targeted ALK therapy. Screening for EML4‐ALK rearrangement by immunohistochemistry should be strongly considered, and next‐generation sequencing is recommended for borderline cases.
Lung cancer is the leading cause of cancer-related mortality worldwide, with non-small cell lung cancer (NSCLC) accounting for over 85% of all cases. Until recently, chemotherapy - characterized by ...some benefit but only rare durable responses - was the only treatment option for patients with NSCLC whose tumors lacked targetable mutations. By contrast, immune checkpoint inhibitors have demonstrated distinctly durable responses and represent the advent of a new treatment approach for patients with NSCLC. Three immune checkpoint inhibitors, pembrolizumab, nivolumab and atezolizumab, are now approved for use in first- and/or second-line settings for selected patients with advanced NSCLC, with promising benefit also seen in patients with stage III NSCLC. Additionally, durvalumab following chemoradiation has been approved for use in patients with locally advanced disease. Due to the distinct features of cancer immunotherapy, and rapid progress in the field, clinical guidance is needed on the use of these agents, including appropriate patient selection, sequencing of therapies, response monitoring, adverse event management, and biomarker testing. The Society for Immunotherapy of Cancer (SITC) convened an expert Task Force charged with developing consensus recommendations on these key issues. Following a systematic process as outlined by the National Academy of Medicine, a literature search and panel voting were used to rate the strength of evidence for each recommendation. This consensus statement provides evidence-based recommendations to help clinicians integrate immune checkpoint inhibitors into the treatment plan for patients with NSCLC. This guidance will be updated following relevant advances in the field.
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